Intracellular communication via ubiquitin (Ub) signaling impacts all aspects of eukaryotic cell biology and regulates pathways critical to human development and viability. Aberrations or defects in Ub-signaling can result in numerous debilitating diseases including neurodegenerative diseases, infections, and cancer. Despite remarkable progress over the decades, we still have only a rudimentary understanding of the molecular factors and networks of interactions that govern the assembly of the proteins required for regulated Ub transfer and signaling. The ability to intervene in diseases related to Ub-signaling requires a thorough understanding of these pathways at the molecular level. The basic scheme for Ub modification involves the concerted activities and interactions of several different proteins. This proposal focuses on expanding our understanding of a central player in Ub-transfer reactions, the class of enzymes known as Ub-Conjugating Enzymes or E2s. Once thought simply to shuttle Ub to the site of modification, emerging evidence suggests that E2s can play a pivotal role in substrate recognition, determining the nature of Ub modification of a target protein, and even interacting with and influencing the activities of proteins outside the traditional Ub-transfer pathway. There are ~40 E2s in the human genome and most are thought to be directly involved in Ub transfer. Very little functional information is available for the majority of these E2s. The Research Plan outlined in this proposal seeks to substantially expand our understanding of E2 function. Using various biochemical approaches, NMR spectroscopy, crystallography, and mass spectrometry, this project seeks to develop a molecular understanding of the factors that govern 1) the intrinsic activity of E2~Ub conjugates toward particular residues, 2) E2~Ub recognition of substrates, and 3) and regulatory interactions of E2~Ub conjugates that regulate proteins outside the main Ub-transfer pathway. We have adapted and developed new tools with which to identify proteins selectively modified by particular E2~Ub conjugates. We make use of a new in coli expression system that can reconstruct Ub-transfer pathways in E. coli. This system allows us to generate particular E2~Ub conjugates and investigate their activity in cells without complications from competing Ub- transfer reactions. The new knowledge we seek to obtain will have a significant impact our understanding of human E2 structure and function and greatly accelerate biological research involving ubiquitylation and ubiquitin signaling.